plants

Article Functional and Genetic Diversity of Bacteria Associated with the Surfaces of Agronomic Plants

Basharat Ali Department of and Molecular Genetics, University of the Punjab, Lahore 54590, Pakistan; [email protected]; Tel.: +92-300-4297937

 Received: 9 February 2019; Accepted: 2 April 2019; Published: 4 April 2019 

Abstract: The main objective of this study was to evaluate the genetic diversity and agricultural significance of bacterial communities associated with the surfaces of selected agronomic plants (carrot, cabbage and turnip). The bacterial diversity of fresh agricultural produce was targeted to identify beneficial plant microflora or opportunistic human pathogens that may be associated with the surfaces of plants. Bacterial strains were screened in vitro for auxin production, biofilm formation and antibiotic resistance. 16S rRNA gene sequencing confirmed the presence of several bacterial genera including Citrobacter, Pseudomonas, Pantoea, , Kluyvera, Lysinibacillus, Acinetobacter, Enterobacter, Serratia, Staphylococcus, Burkholderia, Exiguobacterium, Stenotrophomonas, Arthrobacter and Klebsiella. To address the biosafety issue, the antibiotic susceptibility pattern of strains was determined against different antibiotics. The majority of the strains were resistant to amoxicillin (25 µg) and nalidixic acid (30 µg). Strains were also screened for plant growth-promoting attributes to evaluate their positive interaction with colonized plants. Maximum auxin production was observed with Stenotrophomonas maltophilia MCt-1 (101 µg mL−1) and Bacillus cereus PCt-1 (97 µg mL−1). Arthrobacter nicotianae Lb-41 and Exiguobacterium mexicanum MCb-4 were strong biofilm producers. In conclusion, surfaces of raw vegetables were inhabited by different bacterial genera. Potential human pathogens such as Bacillus cereus, Bacillus anthracis, Enterobacter cloacae, Enterobacter amnigenus and Klebsiella pneumoniae were also isolated, which makes the biosafety of these vegetable a great concern for the local community. Nevertheless, these microbes also harbor beneficial plant growth-promoting traits that indicated their positive interaction with their host plants. In particular, bacterial auxin production may facilitate the growth of agronomic plants under natural conditions. Moreover, biofilm formation may help bacteria to colonize plant surfaces to show positive interactions with host plants.

Keywords: food biosafety; bacterial colonization; antibiotic resistance; biofilm formation; raw-eaten vegetables; bacterial auxin production

1. Introduction Fresh vegetables and fruits are integral components of the human diet and consumed in sufficient amounts to maintain good health [1]. There has been a correlation between foodborne outbreaks and increased production, imports and the consumption of fresh agricultural produce [2]. Raw vegetables can harbor pathogenic microorganisms that can cause serious illnesses to humans after consumption. Routes of microbial contamination include the application of organic wastes to soil as fertilizers, use of fecal-contaminated water for irrigation, direct contact with livestock and post-harvest hygiene issues [3]. In most common practices, fresh fruits and vegetables are consumed raw or processed to minimize the chances of foodborne outbreaks. The frequency of foodborne outbreaks from the consumption of contaminated vegetables and fruits has increased in recent decades. There is a long list of reports that confirms that raw vegetables foster several potentially pathogenic organisms, indicating their key involvement in fresh produce-associated outbreaks [4–9].

Plants 2019, 8, 91; doi:10.3390/plants8040091 www.mdpi.com/journal/plants Plants 2019, 8, 91 2 of 15

True or opportunistic human pathogens have been found and reported with foodborne outbreaks due to their adaptation to soil and plant surfaces [10]. They may be highly competitive for nutrients and produce antimicrobial agents to suppress the native microflora to colonize plant surfaces. For instance, pathogenic strains of and Stenotrophomonas maltophila are well documented to colonize wheat and strawberries, respectively [11]. A similar ability of Burkholderia cepacia to cause infections in plants and humans has also been reported [12]. Besides the true human pathogens ( and enterica), a number of potential human pathogens such as Achromobacter xylosoxidans, Janthinobacterium lividum, Alcaligenes xylosoxidans, Alcaligenes faecalis, Serratia marcescens, , Enterobacter amnigenus, Bacillus cereus, and Stenotrophomonas maltophilia have been reported to be present in plant-associated environments [13,14]. Plant-associated microorganisms can conveniently be categorized in three groups: plant-beneficial, plant pathogenic and opportunistic human pathogenic organisms. The plant-beneficial group of bacteria comprises members of different genera such as Burkholderia, Enterobacter, Stenotrophomonas, Pseudomonas, Herbaspirillum, Ralstonia, Bradyrhizobium, Ochrobactrum, Bacillus, Rhizobium and Staphylococcus. These microorganisms are mostly root-associated and factor in the bivalent interactions with plants and human or animal hosts [15]. The microorganisms of these genera proved themselves beneficial for plants by playing their role in growth promotion, protection from phytopathogens and provision of different elemental nutrients such as nitrogen and phosphorus [16,17]. Many potential pathogens of humans live in close association with plants and, therefore, have adapted to benefit plants in several ways in order to ensure their persistence. They can produce different vital phytohormones such as auxin, gibberellins and cytokinins. Some potentially pathogenic strains of Bacillus and Serratia are noted in the growth promotion of diverse crops such as potato, wheat and maize [18,19]. Therefore, the main objective of the present study was to evaluate the microbiological biosafety and agronomic significance of bacterial species associated with raw vegetables. For this purpose, enriched and selective culture media were used to isolate general and specific bacterial species associated with fresh produce. The final taxonomic status of the bacterial isolates was confirmed by 16S rRNA gene sequencing [20]. Fresh produce-associated microbial hazards have not been uncovered in detail in Pakistan. Therefore, in this study, bacterial diversity associated with carrot, cabbage and turnip was targeted. The main reason to work with these agronomic plants was their poor handling from field to local markets. This may have resulted in their cross contamination with potential human pathogens during transportation. It was also hypothesized that after interacting with host plants, bacterial species may have developed beneficial plant growth-promoting traits. With this in mind, the phylogenetic diversity and agricultural significance of bacterial communities were also inspected to identify the range of bacterial species showing positive interactions with colonized plants.

2. Materials and Methods

2.1. Sample Collection and Culture Media Samples of carrot, cabbage and turnip (12 samples each) were collected from different sites in Lahore, Pakistan. Samples were collected from 12 randomly selected sites that were comprised of small vegetable shops, major vegetable markets and superstores. Samples were collected in sterile plastic bags and brought to the laboratory and processed within 24 h. In order to assess the bacterial diversity, different types of culture media including Luria–Bertani Agar (L-agar), Baird–Parker Agar (BPA), Agar (EMB), (MSA), MacConkey Agar (MAC) and Xylose Deoxycholate Agar (XLD) were used. L-agar is extensively used as a general and non-selective [21]. BPA and MSA are selective for the growth of Gram-positive bacteria specifically for Staphylococcus aureus. EMB, MAC and XLD support the growth of Gram-negative bacteria. EMB agar, in particular, differentiates between lactose and non-lactose fermenter groups of Gram-negative bacteria and is widely used for the of E. coli, Citrobacter freunddii and Enterobacter cloacae. Plants 2019, 8, 91 3 of 15

XLD agar is particularly used for the isolation of Salmonella enterica and flexneri from food and clinical samples [22].

2.2. Isolation of Bacterial Strains Samples were crushed in sterile mortars and pestles and diluted serially before spreading. For serial dilution, 10 g of the crushed sample was added into pre-autoclaved 100 mL normal saline solution (0.85% NaCl) and mixed well. Approximately 50 µL of this dilution was aseptically plated on the surface of L-agar and other selective media as mentioned above. After spreading, the plates were incubated between 30 to 37 ◦C for 24 h. Following incubation, colonies with different morphologies were selected and purified using several rounds of streaking on respective media (Figure S1). The purity of the colonies was also monitored by performing Gram staining.

2.3. 16S rRNA Gene Sequencing For 16S rRNA gene sequencing, genomic DNA was extracted from bacterial cultures grown overnight using a Genomic DNA Purification Kit (Promega, USA). An approximately 1·5-kb DNA fragment containing 16S rRNA gene was amplified using forward 27f and reverse primer 1522r [23]. PCR amplification was performed using 50 µL of Dream TaqTM Green PCR Master Mix (ThermoFisher Scientific, Chelmsford, MA, USA) with 0.5 µg of chromosomal DNA template and 0.5 µM of each primer. The reaction mixtures were incubated in a thermocycler Primus 96 (PeQLab, Erlangen, Germany) at 94 ◦C for 5 min and passed through 30 cycles: denaturation for 20 s at 94 ◦C, primer annealing for 20 s at 50 ◦C and extension at 72 ◦C for 2 min. Final extension was carried out at 72 ◦C for 5 min. The amplified products were purified using a QIAquick Gel Extraction Kit (QIAGEN, Frederick, MD, USA) and the samples were sent to First BASE laboratories (Singapore) for the sequencing of 16S rRNA gene.

2.4. Phylogenetic Analysis The sequences were analyzed using the bioinformatics tool, CHROMAS lite version 2.4.1.0, and homology was searched for in the NCBI Basic Local Alignment Search Tool (BLAST) for the identification of bacterial strains. After identification, all the sequences were aligned with a multiple sequence alignment program (ClustalW) using MEGA 6.05 software [24]. The phylogenetic relationships among different bacterial genera were studied after constructing phylogenetic trees using the neighbor-joining (NJ) method [25].

2.5. Antibiotic Susceptibility Pattern of Bacterial Strains The antibiotic sensitivity pattern of purified bacterial strains was evaluated by following the method of Bauer et al. [26]. Plates of Mueller–Hinton agar were prepared and inoculated with bacterial strains using a sterile cotton swab to ensure confluent growth. The antibiotic susceptibility test discs (Bioanalyse®, Ankara, Turkey) for gentamicin (30 µg), tobramycin (10 µg), amikacin (30 µg), cephalexin (30 µg), chloramphenicol (30 µg), amoxicillin (25 µg), nalidixic acid (30 µg) and tetracycline (30 µg) were aseptically placed to the surface of agar plates at well-spaced intervals. Three sets of plates for each strain or antibiotic were prepared for analysis. The plates were then incubated at 37 ◦C for 24 h. After incubation, the plates were observed for the presence of clear zones of inhibition around the antibiotic disc. Zones were measured in millimeters (mm) using the Inhibition Zone Ruler provided by the manufacturer. The zones were then compared with the standardized chart for antibiotics (M100-S23) given by the clinical laboratory standard institute (CLSI, 2013).

2.6. Functional Diversity of Plant-Associated Bacteria Production of auxin in 25 mL of Luria–Bertani broth (L-broth) medium was detected in the presence of 0 or 500 µg mL−1 of L-tryptophan. Flasks were inoculated with purified bacterial cultures Plants 2019, 8, 91 4 of 15 in triplicate (adjusted to 107 CFUs per mL) and incubated on a shaker in the dark at 37 ◦C for 72 h. After incubation, the cultures were centrifuged (Sigma 1-14, Osterode, Germany) at 18,900 relative centrifugal field (RCF) for 10 min. One milliliter of supernatant was taken in a test tube and 2 mL of Salkowski’s reagent was added [27]. The tubes were kept in the dark for 30 min for the development of pink to red coloration. Approximately 300 µL of each sample was taken from the test tube and added into the wells of a microtiter plate. The intensity of the color was measured at 535 nm using a microplate spectrophotometer (Epoch, BioTek, Winooski, VT, USA). Finally, the concentrations of auxin produced by different isolates were determined by comparing the values with the standard curve. The standard curve was constructed using different concentrations of standard auxin (5 to 200 µg mL−1) in 1 mL of distilled water and processed for colorimetric analysis as mentioned above. Phosphate solubilization ability of the bacterial isolates was determined by streaking strains on Pikovskaya agar medium [28]. Hydrogen cyanide (HCN) activity of bacterial strains was determined as mentioned by Ahmad et al. [29].

2.7. Biofilm Formation To determine the biofilm-forming ability of purified bacterial isolates the method by Christensen et al. [30] was followed with slight modifications. Bacterial strains were streaked on Tryptic Soy Agar (TSA) plates and incubated at 37 ◦C for 24 h. From the TSA plates, the cultures were picked and inoculated in (TSB) medium and incubated as mentioned above. Following incubation, the broth cultures were standardized (optical density adjusted to 0.2 at 600 nm) and 20 µL of each culture was transferred to the wells of a 96-well flat bottom microtiter plate (Orange Scientific) that already contained 180 µL of TSB medium. Negative controls with 200 µL of TSB medium were also kept for comparison. The assay was performed in triplicate for all treatments. To promote biofilm formation, the plates were incubated aerobically on a shaker at 37 ◦C for 72 h. After incubation, the well contents were discarded and washed thrice with 250 µL of sterile distilled water to remove any non-adherent and weakly adherent cells. Plates were air dried for 30 min. The biofilm formed in the wells was fixed with 250 µL/well of 98% methanol for 15 min. After air drying, the fixed bacterial cells were stained with 200 µL of 0.1% v/v crystal violet solution for 5 min. The excessive stain was removed by placing the plate under slow running tap water and the plate was air dried. Re-solubilization of crystal violet with the adherent cells was done by adding 200 µL/well of 33% v/v glacial acetic acid. The optical density (OD) was measured at 570 nm using a microplate spectrophotometer (Epoch, BioTek, Winooski, VT, USA).

2.8. Statistical Analysis Data for bacterial auxin production and biofilm formation was subjected to an analysis of variance (ANOVA) using SPSS 16 program. The means of different treatments were separated using Duncan’s multiple range test (p ≤ 0.05).

3. Results

3.1. 16S rRNA Gene Sequencing The sequences obtained from First BASE laboratories were analyzed and refined by trimming the noise from both ends using the bioinformatics tool, CHROMAS lite version 2.4.1.0 (Sequences Supplementary file included). These sequences were used to search for homology with already identified sequences in NCBI using the default nucleotide Basic Local Alignment Search Tool (BLAST) settings. After comparison, the majority of the strains showed up to 99% similarity with their respective identified species. Analysis showed that the strains belong to Bacillus, Staphylococcus, Exiguobacterium, Lysinibacillus, Arthrobacter, Burkholderia, Acinetobacter, Pseudomonas, Stenotrophomonas, Serratia, Klebsiella, Citrobacter, Enterobacter, Pantoea and Kluyvera Plants 2019, 8, 91 5 of 15 genera. After identification, the sequences were submitted in Genbank under accession numbers KJ865549 to KJ865603 (Tables1–3).

Table 1. 16S rRNA gene sequencing of bacteria isolated from carrot at different temperatures and in different culture media.

Serial Temperature Culture Isolates Identified as Accessions No. for Isolation Media 1 BPc-4 30 ◦C L-agar Bacillus cereus BPc-4 KJ865556 2 EMc-3 37 ◦C L-agar B. anthracis EMc-3 KJ865553 3 LCw-22 30 ◦C L-agar B. cereus LCw-22 KJ865598 4 MSc-5 37 ◦C MSA Staphylococcus warneri MSc-5 KJ865590 5 Xc-7 30 ◦C L-agar Lysinibacillus fusiformis Xc-7 KJ865599 6 BPc-1 37 ◦C MAC Serratia rubidaea BPc-1 KJ865576 7 BPc-3 37 ◦C MAC Pantoea dispersa BPc-3 KJ865552 8 EMc-2 37 ◦C MAC Se. rubidaea EMc-2 KJ865581 9 EMc-4 37 ◦C L-agar Acinetobacter calcoaceticus EMc-4 KJ865567 10 Lc-52 37 ◦C L-agar Ac. calcoaceticus Lc-52 KJ865566 11 Lcr-22 37 ◦C MAC Se. rubidaea Lcr-22 KJ865575 12 Mc-2 37 ◦C MAC Se. rubidaea Mc-2 KJ865602 13 Mc-3 37 ◦C L-agar Stenotrophomonas maltophilia Mc-3 KJ865587 14 Mc-4 37 ◦C L-agar Ac. calcoaceticus Mc-4 KJ865588 15 MSc-1 37 ◦C MAC Pantoea sp. MSc-1 KJ865571 16 Xc-3 37 ◦C EMB Enterobacter cloacae Xc-3 KJ865572 17 Xc-5 30 ◦C L-agar Pseudomonas putida Xc-5 KJ865551 18 Xc-6 37 ◦C EMB Citrobacter freundii Xc-6 KJ865549 Abbreviations: L-agar = Luria–Bertani Agar, MSA = Mannitol Salt Agar, MAC = MacConkey Agar, EMB = Eosine Methylene Blue agar.

Table 2. 16S rRNA gene sequencing of bacteria isolated from cabbage at different temperatures and in different culture media.

Serial Temperature Culture Isolates Identified as Accessions No. for Isolation Media 1 BPb-5 37 ◦C MSA Staph. aureus BPb-5 KJ865591 2 Eb-9 30 ◦C L-agar B. cereus Eb-9 KJ865594 3 Eb-10 30 ◦C L-agar B. thuringiensis Eb-10 KJ865560 4 Lb-41 30 ◦C L-agar Arthrobacter nicotianae Lb-41 KJ865583 5 Lb-61 30 ◦C L-agar B. subtilis Lb-61 KJ865595 6 MCb-3 37 ◦C L-agar Staph. arlettae MCb-3 KJ865592 7 MCb-4 37 ◦C L-agar Exiguobacterium mexicanum MCb-4 KJ865577 8 MCb-6 30 ◦C L-agar B. cereus MCb-6 KJ865559 9 MCb-8 30 ◦C L-agar B. subtilis MCb-8 KJ865584 10 MSb-3 30 ◦C L-agar B. cereus MSb-3 KJ865596 11 MSb-4 37 ◦C L-agar B. anthracis MSb-4 KJ865558 12 Xb-6 30 ◦C L-agar B. anthracis Xb-6 KJ865582 13 BPb-3 37 ◦C L-agar Ac. calcoaceticus BPb-3 KJ865562 14 Eb-1 37 ◦C MAC Klebsiella pneumoniae Eb-1 KJ865601 15 Eb-2 37 ◦C MAC Pa. vagans Eb-2 KJ865561 16 Eb-4 30 ◦C L-agar Ac. calcoaceticus Eb-4 KJ865586 17 Eb-6 30 ◦C L-agar Burkholderia cepacia Eb-6 KJ865578 18 Eb-8 37 ◦C L-agar Ac. calcoaceticus Eb-8 KJ865568 19 Xb-3 37 ◦C MAC Se. rubidaea Xb-3 KJ865564 Abbreviations: L-agar = Luria–Bertani Agar, MSA = Mannitol Salt Agar, MAC = MacConkey Agar, EMB = Eosine Methylene Blue agar. Plants 2019, 8, 91 6 of 15

Table 3. 16S rRNA gene sequencing of bacteria isolated from turnip at different temperatures and in different culture media.

Serial Temperature Culture Isolates Identified as Accessions No. for Isolation Media 1 BPt-5 37 ◦C MSA Staph. equorum BPt-5 KJ865579 2 Lt-41 37 ◦C MSA Staph. xylosus Lt-41 KJ865585 3 Lt-73 37 ◦C MSA Staph. warneri Lt-73 KJ865565 4 MSt-1 37 ◦C MSA Staph. xylosus MSt-1 KJ865600 5 MSt-3 37 ◦C MSA Staph. gallinarum MSt-3 KJ865580 6 MSt-7 30 ◦C L-agar B. cereus MSt-7 KJ865557 7 MSt-8 30 ◦C L-agar B. cereus MSt-8 KJ865589 8 PCt-1 30 ◦C L-agar B. cereus PCt-1 KJ865573 9 Xt-1 37 ◦C MSA Staph. xylosus Xt-1 KJ865597 10 Xt-6 30 ◦C L-agar L. fusiformis Xt-6 KJ865555 11 EMt-1 37 ◦C L-agar Ac. bouvetii EMt-1 KJ865593 12 EMt-5 37 ◦C EMB E. amnigenus EMt-5 KJ865563 13 MCt-1 37 ◦C L-agar St. maltophilia MCt-1 KJ865603 14 MCt-5 37 ◦C MAC Kluyvera cryocrescens MCt-5 KJ865554 15 MCt-6 37 ◦C MAC Se. ureilytica MCt-6 KJ865570 16 MSt-6 37 ◦C L-agar Ac. calcoaceticus MSt-6 KJ865569 17 PCt-2 37 ◦C EMB E. cloacae PCt-2 KJ865574 18 Xt-3 37 ◦C EMB C. werkmannii Xt-3 KJ865550 Abbreviations: L-agar = Luria–Bertani Agar, MSA = Mannitol Salt Agar, MAC = MacConkey Agar, EMB = Eosine Methylene Blue agar.

3.2. Phylogenetic Analysis To study the phylogenetic relationships among bacterial strains, the sequences were first aligned with the multiple sequence alignment program (ClustalW) using MEGA 6 software. The phylogenetic tree was constructed using the neighbor-joining method to show evolutionary relationships among different isolates (Figure1). In the phylogenetic tree, the first cluster was comprised of different strains of Bacillus. The second cluster includes Staphylococcus equorum BPt-5, Staph. gallinarum MSt-3, Staph. warneri MSc-5, Staph. arlettae MCb-3, Staph. xylosus Lt-41, Staph. aureus BPb-5, Staph. xylosus Xt-1, Staph. warneri Lt-73 and Staph. xylosus MSt-1. Similarly, different strains of Acinetobcter (Ac. bouvetii EMt-1, Ac. calcoaceticus MSt-6, Ac. calcoaceticus Mc-4, Ac. calcoaceticus Lc-52, Ac. colcoaceticus BPb-3, Ac. colcoaceticus Eb-4, Ac. colcoaceticus EMc-4, Ac. colcoaceticus Eb-8) covered another large cluster in the tree. Additionally, different strains of Gram-negative genera occupied the lower portion of the phylogenetic tree.

3.3. Bacterial Diversity of Fresh Vegetables The microbiological analysis of carrot showed the association of 18 bacterial strains that belong to 11 bacterial genera (Table1). The highest number for colonization was observed for the genera Serratia, Bacillus and Acinetobacter. For cabbage, 19 bacterial strains that represented nine bacterial genera were detected (Table2). Maximum colonization was shown by the genus Bacillus followed by Acinetobacter and Staphylococcus. Additionally, some strains that specifically colonized cabbage include Ar. nicotianae Lb-41, Ex. mexicanum MCb-4, K. pneumoniae EB-1 and Bur. cepacia Eb-6. In the case of turnip, maximum colonization was shown by the genus Staphyloccous followed by Bacillus and Enterobacter (Table3). Among the 18 strains that were detected from turnip, Kluyvera cryocrescens MCt-5 was specifically associated with this vegetable. Overall, the strains from the genus Bacillus, Staphylococcus, Serratia and Acinetobacter were frequently associated with the surfaces of carrot, turnip and cabbage, whereas, Lysinibacillus, Stenotrophomonas, Citrobacter and Enterobacter were commonly associated with carrot or turnip. Some bacterial genera were found to specifically colonize cabbage (Exiguobacterium, Arthrobacter, Burkholderia and Klebsiella), turnip (Kluyvera) or carrot (Pseudomonas and Pantoea). Plants 2019, 8, 91 7 of 15

Plants 2019, 8, x FOR PEER REVIEW 7 of 16

62 Bacillus cereus MSt-8 KJ865589 96 Bacillus cereus MSb-3 KJ865596 54 Bacillus cereus MSt-7 KJ865557

11 Bacillus cereus BPc-4 KJ865556 54 Bacillus anthracis Xb-6 KJ865582 8 Bacillus subtilis MCb-8 KJ865584 Bacillus cereus MCb-6 KJ865559 Bacillus cereus Eb-9 KJ865594 46 60 11 Bacillus subtilis Lb-61 KJ865595 13 Bacillus cereus LCw-22 KJ865598 Bacillus anthracis EMc-3 KJ865553 Bacillus anthracis MSb-4 KJ865558 46 54 11 Bacillus thuringiensis Eb-10 KJ865560 13 Bacillus cereus PCt-1 KJ865573 Staphylococcus equorum BPt-5 KJ865579 Staphylococcus gallinarum MSt-3 KJ865580 42 19 Staphylococcus warneri MSc-5 KJ865590 84 60 66 Staphylococcus arlettae MCb-3 KJ865592 Staphylococcus xylosus Lt-41 KJ865585 36 Staphylococcus aureus BPb-5 KJ865591 Staphylococcus xylosus Xt-1 KJ865597 91 61 21 Staphylococcus warneri Lt-73 KJ865565 26 Staphylococcus xylosus MSt-1 KJ865600 84 Exiguobacterium mexicanum MCb-4 KJ865577 Lysinibacillus fusiformis Xt-6 KJ865555 38 65 Lysinibacillus fusiformis Xc-7 KJ865599 Arthrobacter nicotianae Lb-41 KJ865583 39 Burkholderia cepacia Eb-6 KJ865578 Acinetobacter bouvetii EMt-1 KJ865593 64 Acinetobacter calcoaceticus MSt-6 KJ865569 57 Acinetobacter calcoaceticus Mc-4 KJ865588 Acinetobacter calcoaceticus Lc-52 KJ865566 59 41 50 Acinetobacter calcoaceticus BPb-3 KJ865562 15 Acinetobacter calcoaceticus Eb-4 KJ865586 9 Acinetobacter calcoaceticus EMc-4 KJ865567 12 63 Acinetobacter calcoaceticus Eb-8 KJ865568 Pseudomonas putida Xc-5 KJ865551 44 Stenotrophomonas maltophilia Mc-3 KJ865587 12 Serratia rubidaea LCr-22 KJ865575

33 Serratia rubidaea EMc-2 KJ865581 47 14 Klebsiella pneumoniae Eb-1 KJ865601 Serratia rubidaea BPc-1 KJ865576 12 45 Serratia rubidaea Mc-2 KJ8655602

14 Serratia rubidaea Xb-3 KJ865564 Pantoea dispersa BPc-3 KJ865552 16 Citrobacter werkmanii Xt-3 KJ865550 43 Citrobacter freundii Xc-6 KJ865549 Enterobacter amnigenus EMt-5 KJ865563 76 16 Enterobacter cloacae Xc-3 KJ865572 99 8 Enterobacter cloacae PCt-2 KJ865574 Pantoea vagans Eb-2 KJ865561 Kluyvera cryocrescens MCt-5 KJ865554 Pantoea sp. MSc-1 KJ865571 Serratia ureilytica MCt-6 KJ865570 82 Stenotrophomonas maltophilia MCt-1 KJ865603

FigureFigure 1. 1.Phylogenetic Phylogenetic analysis analysis of of 55 55 bacterial bacterial isolates associated associated with with the the surfaces surfaces of of fresh fresh vegetables vegetables (carrot, cabbage and turnip). Nucleotide sequences were trimmed after 16S rRNA gene sequencing. (carrot, cabbage and turnip). Nucleotide sequences were trimmed after 16S rRNA gene sequencing. Phylogenies were inferred using the neighbor‐joining method and the tree was constructed using Phylogenies were inferred using the neighbor-joining method and the tree was constructed using MEGA 6 [24]. Numbers at the branch points indicate the percentage of 1000 bootstrap resampling. MEGA 6 [24]. Numbers at the branch points indicate the percentage of 1000 bootstrap resampling.

3.4.3.4. Antibiotic Antibiotic Susceptibility Susceptibility of of Bacterial Bacterial Strains Strains TheThe susceptibility susceptibility of of all all thethe identifiedidentified plant-associatedplant‐associated bacterial bacterial isolates isolates towards towards different different antibioticsantibiotics was was determined. determined. Figure Figure 2 shows2 shows the sensitivity the sensitivity pattern of pattern B. cereus ofLCwB.‐22 cereusand A. calcoaceticusLCw-22 and MSt‐6. Out of 55 strains, 30 and 27 strains were resistant against amoxicillin and nalidixic acid, A. calcoaceticus MSt-6. Out of 55 strains, 30 and 27 strains were resistant against amoxicillin and nalidixic respectively. Both of these drugs are broad‐spectrum antibiotics which are equally effective for Gram‐ acid, respectively. Both of these drugs are broad-spectrum antibiotics which are equally effective for positive and Gram‐negative groups of bacteria (Tables 4 and 5). Resistance against tobramycin, Gram-positive and Gram-negative groups of bacteria (Tables4 and5). Resistance against tobramycin, gentamicin and amikacin were shown by 18, eight and two bacterial strains, respectively. On the gentamicin and amikacin were shown by 18, eight and two bacterial strains, respectively. On the other

Plants 2019, 8, 91 8 of 15 Plants 2019, 8, x FOR PEER REVIEW 8 of 16 hand,other hand, 24 bacterial 24 bacterial strains strains were resistantwere resistant to cephalexin. to cephalexin. Chloramphenicol Chloramphenicol and tetracyclineand tetracycline are also are broad-spectrumalso broad‐spectrum antibiotics antibiotics and resistanceand resistance against against them them was was depicted, depicted, respectively, respectively, by seven by seven and and five strainsfive strains while while others others recorded recorded sensitive sensitive or intermediate or intermediate results. results. For Gram-positive For Gram‐positive bacteria, bacteria, the majority the ofmajority the strains of the recorded strains sensitivity recorded against sensitivity amikacin, against gentamicin, amikacin, tetracycline gentamicin, and chloramphenicoltetracycline and (Tablechloramphenicol4). St. maltophilia (Table Mc-34). St. showed maltophilia the highestMc‐3 showed resistance the against highest the resistance applied antibioticsagainst the as applied out of eight,antibiotics it recorded as out of resistant eight, it against recorded seven resistant antibiotics against (Table seven5). antibiotics Similarly, (TableB. subtilis 5). Similarly,MCb-8, Ent. B. subtilis cloacae PCt-2,MCb‐8,Ent. Ent. amnigenus cloacae PCtEMt-5‐2, Ent. and amnigenusStaph. warneri EMt‐5 andLt-73 Staph. showed warneri resistance Lt‐73 showed for six, five,resistance five and for fivesix, antibiotics,five, five and respectively. five antibiotics, respectively.

Figure 2. Antibiotic susceptibility pattern of purifiedpurified strains of B. cereus LCw-22LCw‐22 and A. calcoaceticuscalcoaceticus MSt-6.MSt‐6. Plates were incubated at 3737 ◦°CC forfor 2424 h.h. Abbreviations:Abbreviations: CC == chloramphenicol,chloramphenicol, NA == nalidixic acid, TE TE = = tetracycline, tetracycline, TOB TOB = =tobramycin, tobramycin, AK AK = amikacin, = amikacin, CN CN= gentamicin, = gentamicin, CL = CLcephalexin, = cephalexin, AX = AXamoxicillin. = amoxicillin.

3.5. Functional Diversity of Bacteria µ −1 Bacterial isolates werewere evaluatedevaluated for for their their auxin auxin production production ability ability in in the the presence presence of of 500 500 μgg mL mL‐ L-tryptophan.1 L‐tryptophan. TheThe highest levels of of auxin auxin were were detected detected for for B.B. cereus cereus PCtPCt-1,‐1, B. B.cereus cereus MSbMSb-3‐3 and and Se. Se.ureilytica ureilytica MCtMCt-6,‐6, which which recorded recorded 5‐, 4 5-,‐ and 4- and4‐fold 4-fold increases, increases, respectively, respectively, compared compared to the to un the‐ un-amendedamended control. control. A significant A significant 3‐fold 3-fold increase increase was was observed observed with with each each of the of thestrains: strains: Pan.Pan. vagans vagans Eb‐ Eb-2,2, Se. Se.rubidaea rubidaea EMcEMc-2,‐2, Ac.Ac. calcoaceticus calcoaceticus EbEb-4,‐4, Se.Se. rubidaea rubidaea McMc-2‐2 and and Cit.Cit. freundii freundii XcXc-6.‐6. The The maximum µ −1 auxin concentrations of of 36, 36, 34 34 and and 33 33 μg gml mL‐I werewere obtained obtained by L. by fusiformisL. fusiformis Xt‐6, Xt-6,Ent. cloacaeEnt. cloacae PCt‐2 PCt-2and K. andpneumoniaeK. pneumoniae Eb‐1, respectively,Eb-1, respectively, in the absence in the of absenceL‐tryptophan. of L-tryptophan. However, in the However, L‐tryptophan in the‐ µ −1 µ −1 L-tryptophan-amendedamended medium, St. maltophilia medium, MCtSt. maltophilia‐1 (101 μg MCt-1mL⁻1), B. (101 cereusg mLPCt‐1), (97B. μ cereusg mLPCt-1⁻1), Se. (97rubidaeag mL Mc‐2), µ −1 µ −1 µ −1 Se.(77 μ rubidaeag mL‐1Mc-2), K. pneumoniae (77 g mL Eb), K.‐1 pneumoniae(75 μg mL⁻Eb-11) and (75 Ent.g mLcloacae) andPCt‐Ent.2 (71 cloacae μg mLPCt-2⁻1) were (71 theg mL most) werepromising the most for in promising vitro auxin for production in vitro auxin (Figure production 3). (Figure3). For HCN production, Pan. dispersa BPc‐3 and B. subtilis Lb‐61 were strongly positive and turned the color of the filter paper to a dark orange (Figure S2). Similarly, Ac. calcoaceticus Eb‐4, Ac.

Plants 2019, 8, 91 9 of 15 Plants 2019, 8, x FOR PEER REVIEW 11 of 16

L‐Tryptophan (0 µg) L‐Tryptophan (500 µg)

120 e e 100 d

) d ‐I 80 d cd c c 60 b a a b b a 40 b Auxin (µg ml ab ab ab a a a 20 a

0 PCT‐1 PCt‐2 MCt‐1 MCt‐5 MSb‐3 Eb‐1 Eb‐2 Eb‐4 EMc‐2 Mc‐2 Xc‐6 Strains

FigureFigure 3. 3. AuxinAuxin productionproduction byby somesome representativerepresentative purifiedpurified bacterialbacterial strainsstrains inin the the presence presence and and absenceabsence of of L-tryptophan. L‐tryptophan. Strains Strains were were grown grown in in Luria–Bertani Luria–Bertani broth broth (L-broth) (L‐broth) at at 37 37◦ °CC for for 72 72 h. h. Bar Bar representsrepresents the the mean mean± ± S.E.S.E. ofof threethree replicates.replicates. DifferentDifferent letters letters on on bars bars indicate indicate significant significant differences differences betweenbetween respective respective treatments treatments using using Duncan’s Duncan’s multiple multiple range range test test ( p(p≤ ≤ 0.05).0.05).

4 Table 4. eAntibiotic susceptibility pattern of different Gram-positive bacterial isolates. For analysis, three3.5 set of plates for each strain or antibiotic were incubated at 37 ◦C for 24 h. 3 Antibiotics 2.5 d d d Strains AK AX CL CN NA TOBc TE C 2 ab abZonesab of Inhibitionab (mm) * ab 1.5 ab a

Biofilm (ʎ=570) a Bacillus cereus BPc-4 18 (S) 10 (R) 8 (R) 14 (I) 14 (I) 15 (S) 22 (S) 10 (R) 1 Staphylococcus equorum BPt-5 26 (S) 24 (R) 11 (R) 20 (S) 0 (R) 16 (S) 16 (I) 26 (S) 0.5 S. aureus BPb-5 18 (S) 8 (R) 18 (S) 18 (S) 14 (I) 16 (S) 26 (S) 24 (S) B. anthracis EMc-3 20 (S) 16 (I) 14 (I) 24 (S) 16 (I) 24 (S) 24 (S) 24 (S) 0 Lb‐41B. cereus MCb‐3Eb-9 MCb‐4 Xt‐1 16 Xt‐3 (S) 20 Eb‐1 (S) Eb‐8 0 (R) LCr‐22 22 (S) Lt‐41 20 (S) LCw‐2212 (R) MCt‐518 Mc‐2 (I) 24 MSb‐4 (S) B. thuringiensis Eb-10 18 (S) 20 (S) 0 (R) 14 (I) 0 (R) 12 (R) 20 (S) 12 (R) Strains S. xylosus Lt-41 20 (S) 24 (R) 22 (S) 20 (S) 0 (R) 18 (S) 22 (S) 20 (S) S. warneri Lt-73 20 (S) 0 (R) 0 (R) 12 (R) 0 (R) 10 (R) 16 (I) 24 (S) FigureArthrobacter 4. Biofilm nicotianae formationLb-41 by some 22 selected (S) 26 purified (S) 16 bacterial (S) 16 strains. (S) 0 Biofilm (R) 14assay (I) was 20 performed (S) 30 (S) in TrypticB. subtilis Soy BrothLb-61 (TSB) medium 28 (S)at 37 20°C. (S) Bar 14represents (I) 30 (S)the mean12 (R) ± S.E.22 (S)of three 26 (S)replicates. 14 (I) DifferentB. cereus lettersLCw-22 on bars indicates significant 16 (I) 14 differences (I) 20 (S) between 16 (S) treatments10 (R) using16 (S) Duncan’s 24 (S) multiple 18 (S) S. arlettae MCb-3 32 (S) 14 (I) 22 (S) 28 (S) 0 (R) 22 (S) 8 (R) 24 (S) range test (p ≤ 0.05). Exiguobacterium mexicanum MCb-4 22 (S) 38 (S) 28 (S) 20 (S) 18 (I) 16 (S) 26 (S) 24 (S) B. cereus MCb-6 20 (S) 14 (I) 18 (S) 16 (S) 0 (R) 14 (I) 24 (S) 16 (I) 4. DiscussionB. subtilis MCb-8 16 (I) 0 (R) 0 (R) 10 (R) 12 (R) 10 (R) 16 (I) 10 (R) S. xylosus MSt-1 24 (S) 22 (R) 10 (R) 20 (S) 0 (R) 20 (S) 20 (S) 24 (S) FreshS. vegetables gallinarum MSt-3 are colonized 14by (I) thousands12 (R) of16 microbial (S) 14 (I) species 0 (R) which10 (R) may 20or (S) may 18not (S) be a part of theirB. natural cereus MSt-7 microbiome. Extraneous 26 (S) 14 microorganisms (I) 32 (S) 24 (S) can be 18 (I)found 18 associated (S) 14 (R) with20 (S)plants due to contaminationB. cereus MSt-8 from different 18 sources (S) 8 (R)such 14as (I)unclean12 (R) irrigation16 (I) water, 18 (S) organic 24 (S) fertilizers, 28 (S) animal andB. human cereus MSb-3 wastes [10]. If pathogenic, 34 (S) 42 (S)these 24 microorganisms (S) 30 (S) 12 can (R) influence18 (S) 24human (S) 30health (S) in B. anthracis MSb-4 14 (I) 16 (I) 12 (I) 14 (I) 14 (I) 20 (S) 18 (I) 20 (S) several ways and thus present serious food safety challenges. A number of studies have already S. warneri MSc-5 24 (S) 40 (S) 38 (S) 30 (S) 14 (I) 24 (S) 28 (S) 24 (S) explored freshB. cereus vegetablesPCt-1 for specific 18 plant (S)‐associated 0 (R) 0 (R) human 14 (I)pathogens 16 (I) [31–33].12 (R) The16 (I)present 16 (I)study demonstratedS. xylosus the biosafetyXt-1 concerns 20 associated (S) 20 (R) with20 raw (S)‐eaten 18 (S) fresh 0 (R)vegetables 18 (S) from 22 (S)some 20 areas (S) of Lahore,Lysinibacillus Pakistan. fusiformisMoreover,Xt-6 little is 16known (I) about 0 (R) the 0 (R)microbiological 14 (I) 14 (I)hazards12 (R) associated18 (I) with 20 (S) fresh agriculturalB. produce anthracis Xb-6in Pakistan. Therefore, 22 (S) in12 the (R) present14 (I) work, 16 (S) we 12focused (R) 16on (S) the sanitation 20 (S) 20 of (S) local L. fusiformis Xc-7 34 (S) 0 (R) 0 (R) 25 (S) 0 (R) 8 (R) 32 (S) 40 (S) vegetable markets with concurrent screening for beneficial bacteria–plant interactions. * Letters in parenthesis indicate the level of sensitivity of the respective antibiotics. Abbreviations: R = resistant, I = intermediate,Although Sno = sensitive. obligate Antibiotics: human AK pathogens = Amikacin; AXwere = Amoxicillin; detected CL in = Cephalexin; our vegetable CN = Gentamicin; samples, NA some potentially= Nalidixic pathogenic acid; TOB = Tobramycin;bacteria that TE =were Tetracycline; isolated C = include Chloramphenicol. B. cereus, B. anthracis, Staph. aurues, Ent. cloacae, Ent. amnigenus and K. Pneumoniae. The presence of such organisms in fresh raw‐eaten vegetables is a serious concern for the consumer’s health. B. cereus is a common foodborne human pathogen that causes food poisoning by producing toxins in food. It has been reported to be present

Plants 2019, 8, 91 10 of 15

Table 5. Antibiotic susceptibility pattern of different Gram-negative bacterial isolates. For analysis, three set of plates for each strain or antibiotic were incubated at 37 ◦C for 24 h.

Antibiotics Strains AK AX CL CN NA TOB TE C Zone of Inhibition (mm) * Serratia rubidaea BPc-1 18 (S) 12 (R) 0 (R) 14 (I) 14 (I) 12 (R) 14 (R) 18 (S) Pantoea dispersa BPc-3 22 (S) 14 (I) 14 (I) 18 (S) 12 (R) 14 (I) 20 (S) 12 (R) Acinetobacter calcoaceticus BPb-3 16 (I) 14 (I) 12 (I) 18 (S) 14 (I) 20 (S) 26 (S) 22 (S) Klebsiella penumoniae Eb-1 18 (S) 0 (R) 14 (I) 18 (S) 18 (I) 12 (R) 20 (S) 22 (S) P. vagans Eb-2 20 (S) 22 (S) 20 (S) 20 (S) 18 (I) 22 (S) 22 (S) 10 (R) A. calcoaceticus Eb-4 20 (S) 0 (R) 10 (R) 12 (R) 14 (I) 16 (S) 18 (I) 20 (S) Burkholderia cepacia Eb-6 24 (S) 16 (I) 20 (S) 18 (S) 0 (R) 20 (S) 20 (S) 14 (I) A. calcoaceticus Eb-8 18 (S) 8 (R) 0 (R) 16 (S) 16 (I) 14 (I) 20 (S) 12 (R) A. bouvetii EMt-1 18 (S) 22 (S) 26 (S) 14 (I) 18 (I) 24 (S) 26 (S) 22 (S) Enterobacter amnigenus EMt-5 18 (S) 0 (R) 0 (R) 12 (R) 12 (R) 10 (R) 16 (I) 22 (S) S. rubidaea EMc-2 26 (S) 12 (R) 16 (S) 16 (S) 0 (R) 14 (I) 22 (S) 16 (I) A. calcoaceticus EMc-4 28 (S) 14 (I) 22 (S) 16 (S) 16 (I) 18 (S) 18 (I) 20 (S) A. calcoaceticus Lc-52 16 (I) 12 (R) 12 (I) 14 (I) 12 (R) 14 (I) 18 (I) 18 (S) S. rubidaea Lcr-22 22 (S) 14 (I) 0 (R) 16 (S) 18 (I) 12 (R) 18 (I) 20 (S) Stenotrophomonas maltophilia MCt-1 16 (I) 0 (R) 0 (R) 14 (I) 22 (S) 10 (R) 20 (S) 22 (S) Kluyvera cryocrescens MCt-5 12 (R) 10 (R) 10 (R) 14 (I) 12 (R) 16 (S) 18 (I) 18 (S) S. ureilytica MCt-6 14 (I) 10 (R) 14 (I) 12 (R) 14 (I) 16 (S) 22 (S) 20 (S) S. rubidaea Mc-2 24 (S) 18 (S) 14 (I) 16 (S) 12 (R) 18 (S) 16 (I) 22 (S) S. maltophilia Mc-3 16 (I) 0 (R) 0 (R) 10 (R) 0 (R) 0 (R) 10 (R) 12 (R) A. calcoaceticus Mc-4 26 (S) 20 (S) 28 (S) 28 (S) 0 (R) 26 (S) 20 (S) 28 (S) A. calcoaceticus MSt-6 28 (S) 26 (S) 24 (S) 28 (S) 0 (R) 24 (S) 28 (S) 24 (S) Pantoea sp. MSc-1 28 (S) 20 (S) 18 (S) 22 (S) 20 (S) 25 (S) 26 (S) 18 (S) E. cloacae PCt-2 12 (R) 14 (I) 10 (R) 10 (R) 0 (R) 10 (R) 18 (I) 18 (S) E. cloacae Xc-3 18 (S) 0 (R) 0 (R) 16 (S) 20 (S) 10 (R) 20 (S) 22 (S) Pseudomonas putida Xc-5 34 (S) 12 (R) 10 (R) 28 (S) 16 (I) 30 (S) 36 (S) 16 (I) Citrobacter freundii Xc-6 18 (S) 10 (R) 0 (R) 16 (S) 14 (I) 10 (R) 14 (R) 20 (S) C. werkmannii Xt-3 16 (I) 0 (R) 0 (R) 18 (S) 16 (I) 12 (R) 18 (I) 20 (S) S. rubidaea Xb-3 22 (S) 12 (R) 0 (R) 18 (S) 22 (S) 14 (I) 16 (I) 22 (S) * Letters in parenthesis indicate level of sensitivity of respective antibiotics. Affiliations: R = resistant, I = intermediate, S = sensitive. Antibiotics: AK = Amikacin; AX = Amoxicillin; CL = Cephalexin; CN = Gentamicin; NA = Nalidixic acid; TOB = Tobramycin; TE = Tetracycline; C = Chloramphenicol.

For HCN production, Pan. dispersa BPc-3 and B. subtilis Lb-61 were strongly positive and turned the color of the filter paper to a dark orange (Figure S2). Similarly, Ac. calcoaceticus Eb-4, Ac. calcoaceticus Eb-8, Ac. calcoaceticus BPb-3, K. pneumoniae Eb-1, B. subtilis MCb-8 and B. cereus MSb-3 produced clear zones after inoculation on Pikovskaya media, which indicated a positive test for phosphate solubilization (Figure S3).

3.6. Biofilm Formation The biofilm-forming potential of purified bacterial isolates was examined by a microtiter plate assay. After 72 h of incubation at 37 ◦C in a 96-well microtiter plate, the cells were stained with crystal violet and the cell biomass was recorded by a spectrophotometer. The strains giving a cell mass OD equal to or above 1 were considered good biofilm producers (Figure4). The bacterial strains Ar. nicotianae Lb-41, Staph. arlettae MCb-3, Ex. mexicanum MCb-4 and Staph. xylosus Xt-1 were observed as strong biofilm producers. Similarly, good biofilm production was also noted with K. pneumoniae Eb-1, Ac. calcoaceticus Eb-8, Se. rubidaea LCr-22, Staph. xylosus Lt-41, B. cereus LCw-22, Kl. ryocrescens MCt-5, Se. rubidaea Mc-2, B. anthracis MSb-4 and Cit. werkmanii Xt-3. The bacterial strains also showed variations in their ability to form an in vitro biofilm. For instance, K. pneumoniae Eb-1, Ac. calcoaceticus Eb-8, B. anthracis MSb-4 and A. nicotianae Lb-41 that were associated with cabbage recorded good potential for biofilm formation as compared to other crop isolates. For carrot, three bacterial strains Plants 2019, 8, x FOR PEER REVIEW 11 of 16

L‐Tryptophan (0 µg) L‐Tryptophan (500 µg)

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0 PCT‐1 PCt‐2 MCt‐1 MCt‐5 MSb‐3 Eb‐1 Eb‐2 Eb‐4 EMc‐2 Mc‐2 Xc‐6 Strains Plants 2019, 8, 91 11 of 15 Figure 3. Auxin production by some representative purified bacterial strains in the presence and absence of L‐tryptophan. Strains were grown in Luria–Bertani broth (L‐broth) at 37 °C for 72 h. Bar that include B. cereus LCw-22 and Se. rubidaea (Mc-2, Lcr-22) showed significant biofilm formation. represents the mean ± S.E. of three replicates. Different letters on bars indicate significant differences In the case of turnip, Staph. xylosus (Lt-41, Xt-1) was found to be very effective as a biofilm producer. between respective treatments using Duncan’s multiple range test (p ≤ 0.05).

4 e 3.5

3

2.5 d d d c 2 ab ab ab ab ab 1.5 ab a

Biofilm (ʎ=570) a 1

0.5

0 Lb‐41 MCb‐3 MCb‐4 Xt‐1 Xt‐3 Eb‐1 Eb‐8 LCr‐22 Lt‐41 LCw‐22 MCt‐5 Mc‐2 MSb‐4 Strains

FigureFigure 4. 4.Biofilm Biofilm formation formation by by some some selected selected purified purified bacterial bacterial strains. strains. Biofilm Biofilm assay assay was was performed performed in Trypticin Tryptic Soy BrothSoy Broth (TSB) (TSB) medium medium at 37 ◦ C.at Bar37 °C. represents Bar represents the mean the± S.E.mean of three± S.E. replicates. of three replicates. Different lettersDifferent on bars letters indicates on bars significant indicates significant differences differences between treatments between treatments using Duncan’s using multiple Duncan’s range multiple test (prange≤ 0.05). test (p ≤ 0.05).

4.4. Discussion Discussion FreshFresh vegetables vegetables are are colonized colonized by by thousands thousands of microbialof microbial species species which which may may or may or may not be not a partbe a ofpart their of naturaltheir natural microbiome. microbiome. Extraneous Extraneous microorganisms microorganisms can be can found be associatedfound associated with plants with due plants to contaminationdue to contamination from different from sourcesdifferent such sources as unclean such as irrigation unclean water, irrigation organic water, fertilizers, organic animal fertilizers, and humananimal wastes and human [10]. If wastes pathogenic, [10]. If these pathogenic, microorganisms these microorganisms can influence can human influence health human in several health ways in andseveral thus ways present and serious thus present food safety serious challenges. food safety A number challenges. of studies A number have of already studies explored have already fresh vegetablesexplored fresh for specific vegetables plant-associated for specific plant human‐associated pathogens human [31– 33pathogens]. The present [31–33]. study The demonstrated present study thedemonstrated biosafety concerns the biosafety associated concerns with raw-eatenassociated fresh with vegetables raw‐eaten fromfresh some vegetables areas of from Lahore, some Pakistan. areas of Moreover,Lahore, Pakistan. little is known Moreover, about little the microbiologicalis known about hazards the microbiological associated with hazards fresh associated agricultural with produce fresh inagricultural Pakistan. Therefore, produce in in Pakistan. the present Therefore, work, wein the focused present on work, the sanitation we focused of localon the vegetable sanitation markets of local withvegetable concurrent markets screening with concurrent for beneficial screening bacteria–plant for beneficial interactions. bacteria–plant interactions. AlthoughAlthough no no obligate obligate human human pathogens pathogens were detectedwere detected in our vegetable in our vegetable samples, some samples, potentially some pathogenicpotentially bacteriapathogenic that bacteria were isolated that were include isolatedB. include cereus, B. B. anthraciscereus, B., Staph.anthracis, aurues Staph., Ent. aurues, cloacae Ent., Ent.cloacae, amnigenus Ent. amnigenusand K. Pneumoniae and K. Pneumoniae.. The presence The ofpresence such organisms of such inorganisms fresh raw-eaten in fresh vegetables raw‐eaten isvegetables a serious concernis a serious for concern the consumer’s for the consumer’s health. B. cereus health.is B. a common cereus is a foodborne common humanfoodborne pathogen human thatpathogen causes that food causes poisoning food poisoning by producing by producing toxins in toxins food. Itin has food. been It has reported been reported to be present to be present in the intestinal tract of mammals and their waste material [34]. B. cereus can cause two distinct types of food poisoning that includes diarrheal or emetic syndrome. Nonhemolytic enterotoxin has been shown to be associated with diarrheal syndrome. It is also responsible for a variety of local and systemic infections [35]. Ent. cloacae, Ent. amnigenus and K. Pneumoniae are members of the family and their incidence in vegetables is also linked to the fecal material of warm-blooded animals. Enterobacter is also a member of the coliform group of bacteria which share their growth properties with human pathogenic bacteria. Their presence clearly indicates the possible presence of pathogens in these vegetables and their consumption without proper precautions can impact human health badly. A study by Falomir et al. [36] also reported the isolation of Ent. cloacae and K. pneumoniae from fresh vegetables. Staphylococcal enterotoxins (SEs) are a major cause of food poisoning, which typically occurs after ingestion of different contaminated food products. It has been shown that SEA and SEH enterotoxins are the most common cause of staphylococcal food poisoning around the world [37]. B. cereus, E. coli, Listeria monocytogenes and Salmonella are considered to be the most frequent bacterial Plants 2019, 8, 91 12 of 15 pathogens associated with fresh produce-related outbreaks [38]. In most of the cases, contaminated vegetables with the members of Enterobacteriaceae were associated with gastrointestinal diseases such as diarrhea [39]. Similarly, produce-related pathogenic organisms such as Clostridium botulinum, B. cereus and S. aureus produce heavy amounts of toxins during their colonization and are the major causes of food poisoning [40]. As the isolation of some potential pathogens from fresh vegetables made the safety of these products questionable, it was necessary to screen all the identified microorganisms for multidrug resistance. High resistances of 52 % (amoxicillin) and 59 % (nalidixic acid) were observed with two broad-spectrum antibiotics used for Gram-positive bacteria (Table4). Antibiotics used against Gram-negative bacteria were effective for the majority of the strains (Table5). However, 57% and 50% resistance was recorded for amoxicillin and cephalexin, respectively. In the present study, a few strains of B. cereus (LCw-22, MSt-7, MSb-3) and Pantoea sp. MSc-1 were sensitive to the tested drugs. This may show the potential of these drugs in bacterial disease prevention. Nevertheless, multiple resistance against amoxicillin, tetracycline, gentamicin, chloamphenicol and other chemotherapeutic agents have been reported in Enterobacter and Klebsiella species isolated from fresh vegetables [36]. In this study, bacterial strains associated with fresh agricultural produce also exhibited beneficial plant growth-promoting attributes; especially, auxin production and biofilm formation. A variety of IAA-producing bacterial strains has been shown to harbor by plants that positively influence plant growth and productivity [41–44]. In the present study, supplementation of L-broth with L-tryptophan enhanced auxin production several folds compared to the control. For instance, Se. rubidaea EMc-2 recorded the lowest production of auxin (12.24 µg mL−1) in the absence of L-tryptophan. However, supplementation of the medium with L-tryptophan resulted in a 2-fold increase in auxin content in liquid culture supernatants (Table S1). L-tryptophan has been considered as precursor for auxin biosynthesis in plants as well as for microbes [20]. The results of present study may indicate the ability of microbes to use the natural source of L-tryptophan from root exudates or soil to produce phytohormones within the plant’s rhizosphere. In this way, microbes may provide an exogenous source of phytohormones to plants, especially during the early stages of development [45]. A variety of bacterial genera can interact and colonize plant surfaces by the formation of biofilms. Biofilm formation on plant surfaces may be associated with symbiotic or pathogenic response depending on the microbial species [46]. Our results showed the colonization of fresh agricultural produce by a few potential human pathogens that belong to the genera of Bacillus, Enterobacter, Staphylococcus and Klebsiella. In present study, K. pneumonia Eb-1 and B. anthracis MSb-1 that were associated with cabbage showed significant biofilm formation (Figure4). Thus, these opportunistic human pathogens can pose severe health hazards for consumers. Agricultural produce may be contaminated by animal droppings, farmworkers’ hands and irrigation water, etc. Fresh produce-related outbreaks can be controlled my managing agricultural and post-harvest practices [47]. The colonization of agronomically important plants by opportunistic human pathogens has been reported. For instance, the presence of B. cereus, P. aeruginosa and S. saprophyticus has been reported within the root system of plants with several plant growth-promoting attributes including IAA [48–50]. It has been reported that pathogenic species to animals and humans are mainly transmitted through the food chain. Therefore, pathogenic bacteria can contaminate plant surfaces and actively interact and colonize them as an alternate host [10]. Moreover, in previous studies, these bacteria have also been shown to exhibit beneficial plant attributes including IAA [51]. Bacterial genera such as Burkholderia, Enterobacter, Stenotrophomonas, Pseudomonas, Bacillus and Staphylococcus are associated with plants and affect their hosts in beneficial ways [11]. They have also been reported to stimulate plant growth by the production of IAA, biofilm formation or phosphate solubilization [20,46]. Thus, bacterial strains showing beneficial traits may be used as biofertilizers to minimize the use or cost of inorganic fertilizers for crop production. Phosphorus (P) is the second most important macronutrient (after nitrogen) required for plant growth and development. Inadequate P availability may cause stunted plant growth, and an abnormal dark green leaf color with reddish to purple tips or margins. Rhizobacteria can secrete organic acids or phosphatases in soils to convert Plants 2019, 8, 91 13 of 15 insoluble P into soluble ions that can be utilized by plants [52]. In the current study, Pan. dispersa BPc-3 and B. subtilis Lb-61 were strongly positive for HCN (Table S2). Production of HCN by bacteria may play a very critical role in the suppression of phytopathogens [49].

5. Conclusions In conclusion, fresh raw vegetables were colonized by a variety of bacterial genera. Although no obligate human pathogens were detected, the presence of a few members of potential human pathogens makes the biosafety of these vegetable questionable. They can cause harmful health effects to consumers. It may be difficult to completely remove these bacteria from food but certain precautionary measures particularly in agricultural practices, harvesting and processing can decrease the number of potential risk factors in these fresh produce products. Nevertheless, due to the close proximity with the plant surfaces, these microbes also harbor beneficial plant growth-promoting traits such as auxin production, mineral solubilization and biofilm formation. Overall, this study reported the association of several bacterial genera from the surfaces of agronomically important raw vegetables. The methodological strategies used in this study will help to investigate or screen general or specific bacterial diversity associated with other raw vegetables. In future, this study can be further extended to identify the indigenous bacterial communities associated with fresh agricultural produce growing in different geographical locations.

Supplementary Materials: The following are available online at http://www.mdpi.com/2223-7747/8/4/91/s1, Figure S1: Purified bacterial colonies on LB agar, Figure S2: HCN production by different purified bacterial isolates, Figure S3: Phosphate solubilization by A. caloaceticus Eb-8 on Pikovskaya medium, Table S1: Auxin production and biofilm formation by plant associated bacteria, Table S2: Phosphate solubilization and HCN production by different bacterial isolates. Author Contributions: B.A. developed the experimental layout, managed lab experiments, performed statistical analysis and wrote this manuscript. Funding: This research received no external funding. Acknowledgments: University of the Punjab, Lahore, Pakistan, is acknowledged for providing financial support for this research work. Conflicts of Interest: The authors declare no conflict of interest.

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